Mycobacterium tuberculosis Infection among 1,659 Silicosis Patients in Zhejiang Province, China

ABSTRACT Silicosis is a well-established risk factor for Mycobacterium tuberculosis infection. This study aimed to estimate the burden and risk factors of M. tuberculosis infection. Silicosis patients from Zhejiang Province were screened for M. tuberculosis by sputum culture, chest radiographs, whole-blood gamma interferon (IFN-γ) release assay (QuantiFERON-TB Gold In-Tube [QFT-GIT]), and tuberculin skin test (TST). Potential risk factors for M. tuberculosis were identified. Data for 1,659 patients were obtained from 1,684 participants. Of these, 1,656 (99.8%) were men, and the average age was 58 (54 to 63) years. The prevalence of active tuberculosis (ATB) was 6,340/100,000 (6.34%) people; the proportion of patients with latent tuberculosis infection (LTBI) was 50.6%. Age (odds ratio [OR] = 1.059, 95% confidence interval [CI] = 1.020 to 1.099, P = 0.003), being underweight (OR = 2.320, 95% CI = 1.057 to 5.089, P = 0.036), and having a history of exposure to TB patients (OR = 4.329, 95% CI = 1.992 to 9.434, P < 0.001) were associated with ATB; BCG vaccination could reduce ATB risk in silicosis patients (OR = 0.541, 95% CI = 0.307 to 0.954, P = 0.034). Among patients without ATB, the QFT-GIT positivity rate was 40.5%, which was affected by silicosis severity, while that of TST was 57.2%. BCG vaccination was an independent factor for LTBI risk reduction (OR = 0.612, 95% CI = 0.468 to 0.801, P < 0.001). The quantitative results of QFT-GIT decreased with silicosis stage (H = 6.037; P = 0.048). In conclusion, M. tuberculosis prevalence was high in silicosis patients. BCG vaccination reduced the risk of both ATB and LTBI in silicosis patients. IMPORTANCE This study evaluated the prevalence of Mycobacterium tuberculosis infection in silicosis patients in mainland China and identified the potential risk factors for both active tuberculosis (ATB) and latent tuberculosis infection (LTBI). We believe that our study makes a significant contribution to the literature because we demonstrated that M. tuberculosis prevalence was high among silicosis patients. BCG vaccination was an independent factor that reduced the risk of M. tuberculosis infection in patients with silicosis. Furthermore, we show that the prevalence of LTBI in patients with silicosis may have been underestimated by immunological detection methods. This study can help to identify targeted subgroups prioritized for M. tuberculosis control and to reduce the risk of disease development.

laten infection is not contagious there is no need for infection control in this group. They provide the prevalence of active TB but give it as the number per 100,000. This is fine if you want to compare the prevalence in these patients to that of the general population but it doesn't give one any idea of the actual proportion of patients in this group with active TB. Better to also give %. And to give overall prevalence of TB in China to demonstrate how this population compares. Were the 349 with a history of TB included in any analyses? They should have probably been included with those with active TB to assess the impact of BCG. Direct comparisons of TST and QFT are problematic. TST picks up nontuberculous mycobacteria infection, BCG and M. tuberculosis infections. QFT is more specific and detects only Mtb. In their analyses using TST, what size did they use? Anyone with a positive reaction or only those with > 10mm? Reactions <10 mm could be due to either BCG or NTM. Assessing BCG vaccination by looking for scars is problematic. Some vaccinated individuals do not produce good scars and scars may fade over time, particularly in those vaccinated at birth who are now in their 50s and 60's. Silicosis is known to be immunosuppressive. Both the TST and the QFT decrease as the silicosis category increases which may indicate immunosuppression due to the disease itself and not a lower risk of TB infection or disease or any protective effect of BCG. Some of the odds ratios are barely over 1.0 and the p values are 0.58, 0.59. Some of the results are barely statistically significant. The Results/Discussion section contains new data? It is not surprising that age was not a significant variable. The age range of this group is very small, 53-64. Multiple other studies have failed to show that BCG at birth protects against adult pulmonary TB and it has never ever been shown to protect against latent TB infection.

Reviewer #2 (Comments for the Author):
In the current manuscript, Yang and colleagues examined medical and demographic characteristics of silicosis patients from China. They found a high prevalence of tuberculosis (both active and latent TB), especially among older individuals who were contacts of TB patients. This study has numerous strengths, including its large study population, detailed methods, and robust analyses. The primary limitation is the absence of a matched control group: e.g., individuals from Zhejiang province who do not have silicosis. Controls would allow a better estimate of the community burden of TB, the relative risk of silicosis, and the protective effect of BCG vaccination.
Minor comments: Lines 250-251: "the prevalence of LTBI in patients with silicosis may have been underestimated" The immunological findings (e.g., lower proportion of lymphocytes at higher stages of silicosis) do support the suggestion that that QFT-GIT may generate false-negative LTBI results. However, others have suggested that BCG vaccination can lead to false-positive TST results (e.g., Lu et al. Clin Infect Dis. 2021. 72:2006-2015.1093/cid/ciaa519). Could BCG status explain the higher proportion of TST positivity (57.2%) compared to QFT-GIT positivity (40.5%)?
Lines 188-189: "old age and low BMI were independent risk factors for ATB". Is the association between BMI and ATB cause or effect? Is lower BMI really a risk factor for developing TB? Or is BMI lower because ATB results in weight loss? In this population, is there any association between type of work and BMI? For example, do tunnellers tend to be smaller than quarry workers?
Lines 74-76: Is the annual number of deaths from silicosis really 4 times the annual number of new cases? Ref 2 is twenty years old. Consider including a newer citation. Mycobacterium tuberculosis is the name of a species which is a group of organisms with shared characteristics. It is not correct to use the term 'Mycobacterium tuberculosis' to refer to the bacterium. Manu authors choose to use the abbreviation MTB (no italics) for 'Mycobacterium tuberculosis bacteria' and then refer to MTB infection.
Lines 148-153. Please clearly define what is meant by QFT-GIT quantitative result. Is it the amount of IFN-gamma in the TB antigen tube minus the amount of IFN-gamma in the nil tube? Did the IFN-gamma response in the mitogen tube vary by age of silicosis stage? Line 175. What TST cut-off was used to determine that 57.2% were TST-positive? Infection with or exposure to non-tuberculosis mycobacteria (NTM) can also generate a positive TST. Were there any cases of NTM infection or disease among the persons studied? Figure 4a. What TST cut-off was used for this Figure? Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.

Dear Prof. Kileen Shier：
Thank you for your letter and the reviewer' comments about our manuscript (Spectrum01451-22). The comments by the reviewer are constructive and helpful for improving the manuscript. We appreciate the general comments from the reviewer "The manuscript is fairly well-written…", "This study has numerous strengths, including its large study population, detailed methods, and robust analyses…". We have modified the manuscript in line with the reviewer's comments. Hereby we submit the revised manuscript for your consideration for publication. We think that we have addressed reviewer's comments to the best degree we could, and we hope this has met the reviewer's and editor's requests. Our detailed point-by-point responses to the comments are as follow:

Reviewer #1 (Comments for the Author):
1) The manuscript is fairly well-written although it is confusing at times to distinguish between the different categories of TB. For example, there is ATB which is active TB,

LTBI which is latent TB infection and then there is Mtb the definition of which is not clear.
The authors need to clearly distinguish in the paper whether they are referring to active TB or latent infection.

Response:
We appreciate this suggestion very much. In our study, M.tb infection is 2)The second sentence of the abstract says they want to ..

."risk factors of Mtb infection and subgroups for infection control". Since laten infection is not contagious there is no need for infection control in this group.
Response: Thanks for your comment. We modified this sentence to "This study aimed to estimate the burden and risk factors of MTB among Chinses silicosis patients." (Line 38).
3)They provide the prevalence of active TB but give it as the number per 100,000. This is fine if you want to compare the prevalence in these patients to that of the general population but it doesn't give one any idea of the actual proportion of patients in this group with active TB. Better to also give %. And to give overall prevalence of TB in China to demonstrate how this population compares.
Response: Thanks for your comment. We have added the percentages of active TB in lines 44,130,176,192 and 274. According to the Global Tuberculosis Report (WHO,2021), the incidence of active TB in China is 58/100,000 and Zhejiang province has a TB incidence of nearly 50/100,000, while the prevalence in silicosis patients is 6,340/100,000 in our study. We have added the data in the revised manuscript.

4)Were the 349 with a history of TB included in any analyses? They should have probably
been included with those with active TB to assess the impact of BCG.

Response:
We appreciate this suggestion very much. We tried to include the previous TB (349) with those with active TB (98) participants, and compared them (447) with those non-TB (479) to assess the impact of BCG. We found that having a BCG scar significantly reduced the risk of TB, with an OR of 0.372 (95% CI 0.281-0.498), P<0.001.
According to our flowchart of study, we include three groups: ATB (98), LTBI (491), and non-TB patients (479) in the analysis. In order to make the study more logical and coherent, we would like to request to show only the results of the BCG comparison of ATB and non-TB.

5)Direct comparisons of TST and QFT are problematic. TST picks up nontuberculous mycobacteria infection, BCG and M. tuberculosis infections. QFT is more specific and detects only Mtb.
Response: We couldn't agree more with you. TST result is interfered by nontuberculous mycobacteria infection and BCG vaccination, but it is more economical and operated easily. QFT is more specific for M. tb. Even so, TST remains an important adjunct to the diagnosis of M. tb infection in developing countries. We believe that comparing QFT and TST can provide more information on the application of TST.

6)In their analyses using TST, what size did they use? Anyone with a positive reaction or
only those with > 10mm? Reactions <10 mm could be due to either BCG or NTM.
Response: Thanks for your question and we agree with the TST reactions <10 mm could be due to either BCG or NTM. As mentioned in the manuscript, the methodology and interpretation standards of TST have been described in our previous work (Line310-311).
A diameter ≥ 10 mm of TST was considered positive as we also pointed out in Line 154.
We had also added the cut-off in the Materials and Methods section to clarify the definition.

7)Assessing BCG vaccination by looking for scars is problematic. Some vaccinated
individuals do not produce good scars and scars may fade over time, particularly in those vaccinated at birth who are now in their 50s and 60s.

Response:
We appreciate your suggestion and agree with you. We did consider this issue in the study design, however, it was very difficult to accurately to recall whether BCG vaccination was inoculated in childhood since our study subjects were 50-60 years old.
After weighing the pros and cons carefully, we thought it was more accurate to use objectively existing scars to judge whether the BCG has been vaccinated. This is one of the limitations in our study. We had added it in Discussion section (Line265-267).

8)Silicosis is known to be immunosuppressive. Both the TST and the QFT decrease as the silicosis category increases which may indicate immunosuppression due to the disease itself and not a lower risk of TB infection or disease or any protective effect of BCG.
Response: Thank you for your opinion and we agree with you very much. As we discussed in Paragraph 5 of the Discussion section, we explored the association between the stages of silicosis and QFT positivity rates as well as quantitative results.
Considering that the QFT detection principle is based on the immune response, the Response: Thank you for your comments. When we explored the risk factors for ATB, we found that ATB was associated with age (P=0.003, adjusted OR=1.059 [95% CI 1.020-1.099]). Since this OR is barely over 1.0, we couldn't draw the conclusion that the age is statistically significant. We had modified that in Line 54, Line 61, Line195-196, and Line 272-273 (Highlighted in manuscript).

10)The Results/Discussion section contains new data?
Response: Thanks for the reminder. We had added the data in Line155-160 in Results section to explore the proportions of leukocytes and lymphocytes among different categories of silicosis.
11) It is not surprising that age was not a significant variable. The age range of this group is very small, 53-64.
Response: Thank you for your comment. Due to the small age range of the study objects, it was really difficult to conclude that whether silicosis patients of all ages have an impact on tuberculosis infection. We could only draw a conclusion that there was no statistical difference between the patients with age group of 53-64 years old.

12)Multiple other studies have failed to show that BCG at birth protects against adult pulmonary TB and it has never ever been shown to protect against latent TB infection.
Response: Thank you for your suggestion. We agree with that BCG at birth may not protects against adult pulmonary TB, as well as LTBI. However, it remains controversial.
Some studies suggested that BCG vaccine had a protective effect in adult decades after vaccination and clarified the casual relationship between BCG vaccine and protection against LTBI [1,2,3]. In our study, BCG vaccination reduced the risk of ATB, with an adjusted OR of 0.541 (95% CI 0.307-0.954) and reduced the risk of LTBI, with an adjusted OR of 0.791 (95% CI 0.692-0.904). Based on our analysis and the previous studies report, we think BCG vaccination in silicosis patients may reduce the TB risk.

Reviewer #2 (Comments for the Author):
1) In the current manuscript, Yang and colleagues examined medical and demographic characteristics of silicosis patients from China. They found a high prevalence of tuberculosis (both active and latent TB), especially among older individuals who were contacts of TB patients. This study has numerous strengths, including its large study population, detailed methods, and robust analyses. The primary limitation is the absence of a matched control group: e.g., individuals from Zhejiang province who do not have silicosis. Controls would allow a better estimate of the community burden of TB, the relative risk of silicosis, and the protective effect of BCG vaccination.
Response: Thank you for your comments. In this study, we focused on the burden and risks of tuberculosis among silicosis patients. It will be better to include individuals from Zhejiang province who do not have silicosis as matched control group, which we will mentioned in the limitation discussion in the revised manuscript (Line269-270).

Minor comments:
2) Lines 250-251: "the prevalence of LTBI in patients with silicosis may have been underestimated". The immunological findings (e.g., lower proportion of lymphocytes at higher stages of silicosis) do support the suggestion that that QFT-GIT may generate false-negative LTBI results. However, others have suggested that BCG vaccination can lead to false-positive TST results (e.g., Lu et al. Clin Infect Dis. 2021. 72:2006-2015 Therefore, in our study, we thought that BCG status couldn't explain the higher proportion of TST positivity compared to QFT-GIT positivity.
3) Lines 188-189: "old age and low BMI were independent risk factors for ATB". Is the association between BMI and ATB cause or effect? Is lower BMI really a risk factor for developing TB? Or is BMI lower because ATB results in weight loss? In this population, is there any association between type of work and BMI? For example, do tunnellers tend to be smaller than quarry workers?
Response: Thank you for your comment. You're right. As a cross-section study, our study cannot draw the conclusion whether lower BMI was a cause or effect of ATB. As we only observed this phenomenon in our study population, we just could draw a conclusion that lower BMI was associated with ATB. We had made modification in revised manuscript.
In this population, BMI wasn't associated between type of work. We compared the BMI between quarry workers and tunnellers (23.

4) Lines 74-76: Is the annual number of deaths from silicosis really 4 times the annual number of new cases? Ref 2 is twenty years old. Consider including a newer citation.
Response: Thank you for your comment. According to the Global Burden of Disease study in 2019, there are more than 120,000 new cases and >7000 deaths annually. Response: Thank you for your comment. We strongly agree with this opinion that TST is interfered by nontuberculous mycobacteria infection. In our study, there weren't any known NTM infection cases among three groups. We examined collection of overnight sputum for the presence of acid-fast bacilli by smear (Ziehl-Nielsen technique) and mycobacteria culture, then those who had positive NTM results didn't undergo the immunological tests (QFT or TST). Therefore, NTM patients were not included in our analysis. We had added this information in Line 104-105. Figure 4a. What TST cut-off was used for this Figure? Response: Thank you for your comment. A diameter ≥ 10 mm was considered positive.

5)
We have revised the manuscript in line with all the reviewer's comments and we hope that the manuscript is now acceptable for publication at Microbiology Spectrum. If you have any questions, please feel free to contact us. We appreciate your support very much. Hereby we submit the revised manuscript for your consideration for publication. We think that we have addressed your comments to the best degree we could, and we hope this has met your requests. Our detailed point-by-point responses to the comments are as follow:

This manuscript found four (4) correlations between risk factors in silicosis patients
and active TB:

•BMI, but unclear whether this is a cause or a result of TB infection
•Patients with a known exposure to TB are more likely to have active TB (this is obvious) •Age, but the age group size was small (53-64) and the correlation was not statistically significant

•BCG vaccination, which is the only meaningful correlation based upon the data presented in this manuscript
The study is interesting because of the unique population and study size of silicosis patients. However, the conclusions drawn for the first three points above lack supporting data. I would be willing to consider a revised manuscript with emphasis on the BCG vaccination. Alternatively, the authors may consider submitting to another journal that will accept the manuscript as is.
Response: Thanks for your suggestion, we really appreciate your positive feedback and we agree with you that we didn't know the cause-and-effect relationship between BMI and TB infection thus we have removed BMI as a risk factor in the discussion part. Also, it's apparent that close contact with ATB patients will increase the risk of developing TB, therefore, we didn't underline it anymore. In addition, considering age as a risk factor also lacking strong evidence since the age group size was small, we have emphasized on the BCG vaccination in our revised manuscript and draw a conclusion that the BCG vaccination was an independent factor that reduced the risk of MTB in patients with silicosis. As marked in 52-54, 60-61, 174-176, 276-279 we removed the age and low BMI from the conclusion and in Line 196-203, we revised as "It is well-known that contacts of people with tuberculosis are at high risk of also developing tuberculosis. Since the age group size in our study was small and the correlation was not statistically significant, it was difficult to consider age as a risk factor. Meanwhile, in the cross-sectional study, whether low BMI was a cause or a result of TB infection was not known.……It was worth noting that BCG vaccination proved to be a protective factor against ATB". In addition, since we no longer emphasize the factors of age and weight, we have deleted Figure 2 to make the article clearer. This comment is helpful for